Variant Classification for Pompe disease; ACMG/AMP specifications from the ClinGen Lysosomal Diseases Variant Curation Expert Panel.

Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.

Mechanistic Investigation of GHS-R Mediated Glucose-Stimulated Insulin Secretion in Pancreatic Islets.

Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), is expressed in the pancreas. Emerging evidence indicates that GHS-R is involved in the regulation of glucose-stimulated insulin secretion (GSIS), but the mechanism by which GHS-R regulates GSIS in the pancreas is unclear. In this study, we investigated the role of GHS-R on GSIS in detail using global Ghsr-/- mice (in vivo) and Ghsr-ablated pancreatic islets (ex vivo). GSIS was attenuated in both Ghsr-/- mice and Ghsr-ablated islets, while the islet morphology was similar between WT and Ghsr-/- mice. To elucidate the mechanism underpinning Ghsr-mediated GSIS, we investigated the key steps of the GSIS signaling cascade. The gene expression of glucose transporter 2 (Glut2) and the glucose-metabolic intermediate-glucose-6-phosphate (G6P) were reduced in Ghsr-ablated islets, supporting decreased glucose uptake. There was no difference in mitochondrial DNA content in the islets of WT and Ghsr-/- mice, but the ATP/ADP ratio in Ghsr-/- islets was significantly lower than that of WT islets. Moreover, the expression of pancreatic and duodenal homeobox 1 (Pdx1), as well as insulin signaling genes of insulin receptor (IR) and insulin receptor substrates 1 and 2 (IRS1/IRS2), was downregulated in Ghsr-/- islets. Akt is the key mediator of the insulin signaling cascade. Concurrently, Akt phosphorylation was reduced in the pancreas of Ghsr-/- mice under both insulin-stimulated and homeostatic conditions. These findings demonstrate that GHS-R ablation affects key components of the insulin signaling pathway in the pancreas, suggesting the existence of a cross-talk between GHS-R and the insulin signaling pathway in pancreatic islets, and GHS-R likely regulates GSIS via the Akt-Pdx1-GLUT2 pathway.

Untargeted metabolomics as an unbiased approach to the diagnosis of inborn errors of metabolism of the non-oxidative branch of the pentose phosphate pathway.

Inborn errors of metabolism (IEM) involving the non-oxidative pentose phosphate pathway (PPP) include the two relatively rare conditions, transketolase deficiency and transaldolase deficiency, both of which can be difficult to diagnosis given their non-specific clinical presentations. Current biochemical testing approaches require an index of suspicion to consider targeted urine polyol testing. To determine whether a broad-spectrum biochemical test could accurately identify a specific metabolic pattern defining IEMs of the non-oxidative PPP, we employed the use of clinical metabolomic profiling as an unbiased novel approach to diagnosis. Subjects with molecularly confirmed IEMs of the PPP were included in this study. Targeted quantitative analysis of polyols in urine and plasma samples was accomplished with chromatography and mass spectrometry. Semi-quantitative unbiased metabolomic analysis of urine and plasma samples was achieved by assessing small molecules via liquid chromatography and high-resolution mass spectrometry. Results from untargeted and targeted analyses were then compared and analyzed for diagnostic acuity. Two siblings with transketolase (TKT) deficiency and three unrelated individuals with transaldolase (TALDO) deficiency were identified for inclusion in the study. For both IEMs, targeted polyol testing and untargeted metabolomic testing on urine and/or plasma samples identified typical perturbations of the respective disorder. Additionally, untargeted metabolomic testing revealed elevations in other PPP metabolites not typically measured with targeted polyol testing, including ribonate, ribose, and erythronate for TKT deficiency and ribonate, erythronate, and sedoheptulose 7-phosphate in TALDO deficiency. Non-PPP alternations were also noted involving tryptophan, purine, and pyrimidine metabolism for both TKT and TALDO deficient patients. Targeted polyol testing and untargeted metabolomic testing methods were both able to identify specific biochemical patterns indicative of TKT and TALDO deficiency in both plasma and urine samples. In addition, untargeted metabolomics was able to identify novel biomarkers, thereby expanding the current knowledge of both conditions and providing further insight into potential underlying pathophysiological mechanisms. Furthermore, untargeted metabolomic testing offers the advantage of having a single effective biochemical screening test for identification of rare IEMs, like TKT and TALDO deficiencies, that may otherwise go undiagnosed due to their generally non-specific clinical presentations.

Impaired mitochondrial complex I function as a candidate driver in the biological stress response and a concomitant stress-induced brain metabolic reprogramming in male mice.

Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi-platform targeted mass spectrometry-based metabolomics. Ndufs4GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4GT/GT from WT mice. Specifically, we observed a reversed TCA cycle flux and rewiring of amino acid metabolism in the prefrontal cortex. Next, exposing mice to chronic variable stress (a model for depression-like behavior), we found that Ndufs4GT/GT mice showed altered stress response and coping strategies with a robust stress-associated reprogramming of amino acid metabolism. Our data suggest that impaired mitochondrial CI function is a candidate driver for altered stress reactivity and stress-induced brain metabolic reprogramming. These changes result in unique phenomic and metabolomic signatures distinguishing groups based on their mitochondrial genotype.

Sphingolipid Metabolism Perturbations in Rett Syndrome.

Rett syndrome is a severe neurodevelopmental disorder affecting mostly females and is caused by loss-of-function mutations in the MECP2 gene that encoded the methyl-CpG-binding protein 2. The pathogenetic mechanisms of Rett syndrome are not completely understood and metabolic derangements are emerging as features of Rett syndrome. We performed a semi-quantitative tandem mass spectrometry-based analysis that measured over 900 metabolites on blood samples from 14 female subjects with Rett syndrome carrying MECP2 mutations. The metabolic profiling revealed alterations in lipids, mostly involved in sphingolipid metabolism, and sphinganine/sphingosine, that are known to have a neurotrophic role. Further investigations are required to understand the mechanisms underlying such perturbations and their significance in the disease pathogenesis. Nevertheless, these metabolites are attractive for studies on the disease pathogenesis and as potential disease biomarkers.

2-Pyrrolidinone and Succinimide as Clinical Screening Biomarkers for GABA-Transaminase Deficiency: Anti-seizure Medications Impact Accurate Diagnosis.

Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-aminobutyrate aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.

Opening a window on lysosomal acid lipase deficiency: Biochemical, molecular, and epidemiological insights.

Lysosomal acid lipase deficiency (LAL-D) is a multi-organ autosomal recessive disease caused by mutations in LIPA. We reviewed data from 681 samples (white blood cells [WBC] n = 625, fibroblasts = 30, liver = 4, amniocytes = 13, chorionic villus = 9) received for analysis of lysosomal acid lipase (LAL) activity over a 15-year period. LIPA sequencing was performed in 49 patients with reduced (n = 26) or deficient (n = 23) LAL activity. The Exome Aggregation Consortium and Genome Aggregation Database dataset were used for LAL-D prevalence calculations. LAL WBC activity was reduced in 67 patients (10.72%) and deficient in 37 (5.92%). The average of LAL activity ± margin of error (CI 95%) was 19.32 ± 0.86 pmol/min/mg for reduced activity patients and 5.90 ± 1.42 pmol/min/mg for deficient patients. The average age at diagnosis for LAL-D was 23.6 years with several patients older than age 30. The correlation between the age at diagnosis and LAL activity showed a significant moderate direct correlation (Pearson's r = 0.46, P < 0.005). Homozygous or compound heterozygous mutations were identified in 9 out of 23 patients with deficient results (detection rate 39.1%). The average LAL activity in molecularly confirmed patients was 4.02 ± 2.02 pmol/min/mg protein, while in molecularly negative patients was 13.886 ± 1.49 pmol/min/mg (P < 0.0001). Twenty-two different mutations were identified including two novel variants (c.309C>A and c.856G>C). A carrier frequency of approximately 1 in 350 was inferred. LAL activity in WBC is a validated tool for LAL-D diagnosis. Higher residual enzymatic activity might result in a milder phenotype leading to diagnosis delay. A cut-off below 12 pmol/min/mg protein might be useful to discriminate patients with LIPA mutations.

Corrigendum: 2-Pyrrolidinone and Succinimide as Clinical Screening Biomarkers for GABA-Transaminase Deficiency: Anti-seizure Medications Impact Accurate Diagnosis.

[This corrects the article DOI: 10.3389/fnins.2019.00394.].

Correction: Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress.

[This corrects the article DOI: 10.1371/journal.pbio.1002197.].

A metabolomic map of Zellweger spectrum disorders reveals novel disease biomarkers.

PURPOSE: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown. METHODS: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds. RESULTS: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes. CONCLUSION: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.

Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet.

Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS) is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF)/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.

Analyses of SLC13A5-epilepsy patients reveal perturbations of TCA cycle.

OBJECTIVE: To interrogate the metabolic profile of five subjects from three families with rare, nonsense and missense mutations in SLC13A5 and Early Infantile Epileptic Encephalopathies (EIEE) characterized by severe, neonatal onset seizures, psychomotor retardation and global developmental delay. METHODS: Mass spectrometry of plasma, CSF and urine was used to identify consistently dysregulated analytes in our subjects. RESULTS: Distinctive elevations of citrate and dysregulation of citric acid cycle intermediates, supporting the hypothesis that loss of SLC13A5 function alters tricarboxylic acid cycle (TCA) metabolism and may disrupt metabolic compartmentation in the brain. SIGNIFICANCE: Our results indicate that analysis of plasma citrate and other TCA analytes in SLC13A5 deficient patients define a diagnostic metabolic signature that can aid in diagnosing children with this disease.

Mitochondrial Dysfunction Mediated by Poly(ADP-Ribose) Polymerase-1 Activation Contributes to Hippocampal Neuronal Damage Following Status Epilepticus.

Mitochondrial dysfunction plays a central role in the neuropathology associated with status epilepticus (SE) and is implicated in the development of epilepsy. While excitotoxic mechanisms are well-known mediators affecting mitochondrial health following SE, whether hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) also contributes to SE-induced mitochondrial dysfunction remains to be examined. Here we first evaluated the temporal evolution of poly-ADP-ribosylated protein levels in hippocampus following kainic acid-induced SE as a marker for PARP-1 activity, and found that PARP-1 was hyperactive at 24 h following SE. We evaluated oxidative metabolism and found decreased NAD⁺ levels by enzymatic cycling, and impaired NAD⁺-dependent mitochondrial respiration as measured by polarography at 24 h following SE. Stereological estimation showed significant cell loss in the hippocampal CA1 and CA3 subregions 72 h following SE. PARP-1 inhibition using N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)- N,N-dimethylacetamide (PJ-34) in vivo administration was associated with preserved NAD⁺ levels and NAD⁺-dependent mitochondrial respiration, and improved CA1 neuronal survival. These findings suggest that PARP-1 hyperactivation contributes to SE-associated mitochondrial dysfunction and CA1 hippocampal damage. The deleterious effects of PARP-1 hyperactivation on mitochondrial respiration are in part mediated through intracellular NAD⁺ depletion. Therefore, modulating PARP-1 activity may represent a potential therapeutic target to preserve intracellular energetics and mitochondrial function following SE.

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse.

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.

Elucidation of the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profiling assay.

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.

Homozygous variants in pyrroline-5-carboxylate reductase 2 (PYCR2) in patients with progressive microcephaly and hypomyelinating leukodystrophy.

Pyrroline-5-carboxylate reductase 2, encoded by PYCR2, is one of the three homologous enzymes that catalyze the last step of proline synthesis. Homozygous variants in PYCR2 have been reported in patients from multiple consanguineous families with hypomyelinating leukodystrophy 10 (HLD10) (MIM: 616420). Here, we report five additional patients from three families with homozygous nonsense or missense variants in PYCR2, identified through clinical exome sequencing. All patients presented with postnatally acquired microcephaly, moderate to profound global developmental delay, and failure to thrive. Brain MRI in these patients showed thin corpus callosum, delayed myelination, and generalized white-matter volume loss. Additional phenotypes that were less consistent among patients included seizures or seizure-like movements, spasticity and ataxic gait, recurrent vomiting, cortical blindness, dysmorphic features, joint contractures, and irritability. Exome sequencing identified homozygous variants in PYCR2 in the proband from each family: c.28C>T (p.(Glu10Ter)), c.796C>T (p.(Arg266Ter)), and c.577G>A (p.(Val193Met)). Subsequent targeted analyses demonstrated co-segregation of the disease with the variant in the family. Despite the metabolic role of PYCR2, routine serum metabolic test in these patients were normal. To further understand the disease etiology and functions of PYCR2, small molecule metabolomics profiling was performed in plasma from three severely affected patients. No significant changes were identified in proline biosynthesis pathway or related metabolites. Studying the clinical features and the metabolic profiles of the PYCR2-deficient patients provides a more comprehensive picture for this newly identified disorder and facilitates further research on the gene function and disease etiology. © 2016 Wiley Periodicals, Inc.

Expansion of the Phenotypic Spectrum of Propionic Acidemia with Isolated Elevated Propionylcarnitine.

We report three patients with elevations of propionylcarnitine (C3), one without elevations of 2-methylcitrate and 3-hydroxypropionate in urine organic acid analysis, and the other two showing only mild elevations, all of whom were subsequently confirmed to have propionic acidemia by molecular analysis of PCCA and PCCB genes. To date, they have had a mild clinical course. These cases illustrate the importance of considering high C3 as the only biochemical abnormality in a diagnosis of propionic acidemia. Since mild C3 elevations may be overlooked and considered non-diagnostic in isolation, we advise considering a diagnosis of propionic acidemia even in the absence of significant elevations 2-methylcitrate or 3-hydroxypropionate in urine organic acid analysis.

Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration.

We previously identified mutations in Nardilysin (dNrd1) in a forward genetic screen designed to isolate genes whose loss causes neurodegeneration in Drosophila photoreceptor neurons. Here we show that NRD1 is localized to mitochondria, where it recruits mitochondrial chaperones and assists in the folding of α-ketoglutarate dehydrogenase (OGDH), a rate-limiting enzyme in the Krebs cycle. Loss of Nrd1 or Ogdh leads to an increase in α-ketoglutarate, a substrate for OGDH, which in turn leads to mTORC1 activation and a subsequent reduction in autophagy. Inhibition of mTOR activity by rapamycin or partially restoring autophagy delays neurodegeneration in dNrd1 mutant flies. In summary, this study reveals a novel role for NRD1 as a mitochondrial co-chaperone for OGDH and provides a mechanistic link between mitochondrial metabolic dysfunction, mTORC1 signaling, and impaired autophagy in neurodegeneration.

Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado) in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs) has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes.

Expanding the phenotypic spectrum of Succinyl-CoA ligase deficiency through functional validation of a new SUCLG1 variant.

Deficiency of the TCA cycle enzyme Succinyl-CoA Synthetase/Ligase (SCS), due to pathogenic variants in subunits encoded by SUCLG1 and SUCLA2, causes mitochondrial encephalomyopathy, methylmalonic acidemia, and mitochondrial DNA (mtDNA) depletion. In this study, we report an 11year old patient who presented with truncal ataxia, chorea, hypotonia, bilateral sensorineural hearing loss and preserved cognition. Whole exome sequencing identified a heterozygous known pathogenic variant and a heterozygous novel missense variant of uncertain clinical significance (VUS) in SUCLG1. To validate the suspected pathogenicity of the novel VUS, molecular and biochemical analyses were performed using primary skin fibroblasts from the patient. The patient's cells lack the SUCLG1 protein, with significantly reduced levels of SUCLA2 and SUCLG2 protein. This leads to essentially undetectable SCS enzyme activity, mtDNA depletion, and cellular respiration defects. These abnormal phenotypes are rescued upon ectopic expression of wild-type SUCLG1 in the patient's fibroblasts, thus functionally confirming the pathogenic nature of the SUCLG1 VUS identified in this patient and expanding the phenotypic spectrum for SUCLG1 deficiency.